Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P35/00—Preparation of compounds having a 5-thia-1-azabicyclo [4.2.0] octane ring system, e.g. cephalosporin
  • C12P35/08—Preparation of compounds having a 5-thia-1-azabicyclo [4.2.0] octane ring system, e.g. cephalosporin disubstituted in the 7 position
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/14—Fungi; Culture media therefor
  • C12N1/16—Yeasts; Culture media therefor
  • C12N1/165—Yeast isolates
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/645—Fungi ; Processes using fungi
  • C12R2001/88—Torulopsis

Definitions

• the present invention relates to a process for producing 7 ⁇ -(D-5-­ amino-5-carboxyvaleramido)-3-hydroxymethyl-7 ⁇ -methoxy-3-cephem-4-­ carboxylic acid (I) (hereafter simply referred to as "oganomycin E”).
• the present invention provides a process for producing oganomycin E which comprises allowing yeast belonging to the genus Torulopsis , or esterase derived from the yeast to act on cephamycin compound (II) of general formula : wherein R1 represents a hydrogen atom or a methoxy group and R2 represents a hydroxy group or a sulfoxy group; and to a process for producing oganomycin E (I) which comprises culturing cephamycin compound (II)-producing bacteria belonging to the genus Streptomyces and allowing yeast belonging to the genus Torulopsis , or esterase derived from the yeast to act on accumulated cephamycin compound (II) in the culture solution to form oganomycin E (I).
• the invention may provide oganomycin E in salt form; such salts are useful in the field of cephalosporins.
• the yeast or esterase derived therefrom may be on a substrate, i.e. bound on a solid carrier.
• Known methods for producing oganomycin E by fermentation include culturing Streptomyces chartriusis SF-1623 under aerobic conditions and harvesting this substance from the culture solution (Published Unexamined Japanese Patent Application No.121488/75) and culturing Streptomyces oganonensis and harvesting oganomycin E from the culture solution (Published Unexamined Japanese Patent Application No.43697/82).
• the former method provides a low yield and is unsuited for industrial production.
• the latter method gives at least 1000 times the yield but the concentration of oganomycin E accumulated in the culture solution is approximately 5 mg/ml.
• Cephamycin compounds (II) may be by-produced during production of oganomycin E by fermentation culturing of oganomycin E-producing bacteria.
• the present invention can provide an improved yield of oganomycin E by conversion of the cephamycin compounds (II) by-produced by oganomycin E-producing bacteria.
• the present invention can also provide a process for production of oganomycin E using bacteria capable of producing cephamycin compounds (II).
• oganomycin E-producing bacteria examples include Streptomyces chartriusis SF-1623 (Published Unexamined Japanese Patent Application No. 121488/75) and Streptomyces oganonensis Y-G19Z (Published Unexamined Japanese Patent Application No.79394/80 and Japanese Patent Application No.110857/85) described above.
• Examples of bacteria capable of producing cephamycin compounds (II) include Streptomyces griceus MA-2837 and MA-4125a (Published Unexamined Japanese Patent Application No.3286/71), Streptomyces viridochromogenes, Streptomyces fimbriatus, Streptomyces halstedii, Streptomyces rochei, Streptomyces cinnamonensis and Streptomyces chartrensis (Belgian Patent 764,160). Further the aforesaid Streptomyces oganonensis Y-G19Z by produces cephamycin compounds (II) and thus can also be cephamycin compound-producing bacteria.
• the esterase derived from the yeasts belonging to the genus Torulopsis which can be used in the present invention is an enzyme capable of hydrolyzing the ester bond at the 3-side chain of the aforesaid cephamycin compounds (II).
• the esterase may be bound to a solid carrier or substrate.
• a material containing the esterase may be used in the invention; examples of such material are microorganisms carrying the esterase, immobilized esterase etc.
• Vegetative cells are spherical or oval, sometimes protractile.
• the size is 3.0 to 10 ⁇ m x 2.0 to 5 ⁇ m with oval cells and 3 to 11 ⁇ m with spherical ones.
• Vegetative propagation occurs by multipolar budding. Pseudomycel, chlamydospore, oidispore, budding spore, etc. formations are absent but a trace of pseudomycel-like formation sometimes occurs. Neither ascospores nor balistospores are formed.
• the medium is somewhat turbid as a whole and colored pale pink to light orange. Neither skin nor precipitate is formed. Generation of gas is not observed.
• Colonies are smooth, glossy, colored pale orange to light orange and rise to a hemispherical shape on agar. No diffusible dye is formed.
• Fermentation of glucose is weak but positive. Fermentation of fructose, galactose, sucrose, maltose and raffinose is negative.
• Negative bacterial dye is a non-carotinoidal sub­ stance insoluble in acetone and petroleum ether
• this strain belongs to a non-spored yeast; nutrient cells are spherical or oval; and pseudo-hyphae are sometimes observed as traces; no starch-like substance is formed but assimilation of nitrates is positive; neither skin nor precipitate is formed by liquid culture; colonies are colored pale orange to light orange but this dye is not of carotinoid; further fermentation of glucose is weak but positive.
• genera of micro­ organisms having these properties are the genus Cryptococcus and the genus Torulopsis . In Cryptoccus, cells are covered with a capsule, colonies are viscous and starch-like substance is formed. This strain does not show such properties and hence is distinguishable. The morphological properties, physiological properties, etc.
• the new strain has characteristic enzymic esterase activity and has been named as the type strain Torulopsis sp . YE-0807L.
• the type strain has been deposited in the Agency of Industrial Science and Technology, the Fermentation Research Institute under the accession number FERM BP-1158.
• microorganisms When selecting a fermentation metabolism system particular attention should be paid to avoiding inhibition of the system and achieving the optimum conditions. This is particularly important in the case of adopting a so-called mixing culture method in which several microorganisms are co-cultured - e.g. cephamycin compound-producing bacteria are cultured in the presence of esterase-producing yeast to produce oganomycin E.
• Some microorganisms may have a potent productivity of amylases or proteases, they may have a potent productivity of acidic substances or basic substances, they may have a large propagation rate or oxygen absorption rate, etc., and they may disrupt the fermentation metabolism environment and hence reduce the total product yield.
• yeasts are a good group of microorganisms as compared to, e.g. bacteria and molds.
• oganomycin E can be prepared as follows :
• This process comprises using cephamycin compound (II) as a substrate, and allowing yeast belonging to the genus Torulopsis , or the esterase produced therefrom, to act thereon to produce oganomycin E.
• yeast belonging to the genus Torulopsis , or the esterase produced therefrom (which may or may not be carried on a substrate)
• a solution of the cephamycin compound (II) is mixed with a solution of the cephamycin compound (II); the mixture is shaken at about 30°C at neutral pH and the formed oganomycin E is separated.
• the solution of the cephamycin compound (II) there may be used any of a fermentation solution containing the compound (II), a fermentation filtrate and a solution of the compound (II) separated and isolated.
• the esterase source there can be utilized the culture solution per se of the yeast belonging to the genus Torulopsis , cells of the yeast, ground cells, an extract of the esterase active fraction, solid carriers (activated charcoal, diatomaceous earth, hydrophilic gel, high molecular resins, etc.) having immobilized thereon yeast belonging to the genus Torulopsis or esterase derived therefrom.
• solid carriers activated charcoal, diatomaceous earth, hydrophilic gel, high molecular resins, etc.
• cephamycin compound-producing strain is cultured under culture conditions used for the production of the cephamycin compound by fermentation and yeast belonging to the genus Torulopsis is inoculated and both strains are simultaneously cultured.
• deep culture using liquid medium is advantageous.
• the esterase acts in a bacteria-free environment.
• the medium used for the mixing incubation may be any medium as far as it contains nutrient sources that the cephamycin compound-producing bacteria,e.g. those belonging to the genus Streptomyces , can utilize.
• synthetic medium, semi-synthetic medium or natural medium can be used.
• glucose, sucrose, mannitol, glycerine, dextrin, starch, vegetable oils, etc. are used as carbon sources and as nitrogen sources, meat extract, peptone, gluten meal, cotton seed lees, soybean powders, peanut powders, fish powders, corn steep liquor, dry yeast, yeast extract, ammonium sulfate, ammonium nitrate, urea and other organic or inorganic nitrogen sources are employed.
• metal salts e.g. sulfates, nitrates, chlorides, carbonates, phosphates, etc. of Na, K, Mg, Ca, Zn, Fe, etc.
• the incorporation of magnesium carbonate is effective for increasing the productivity (titer) of the compound (I).
• appropriate antibiotic production-accelerating substances or defoam­ ing agents such as methionine, cystein, cystine, methyl oleate, lard oil, silicone oil, surfactants, etc.may be used.
• the culturing temperature be in a range of about 18 to about 35°C, preferably about 30°C. Good results are obtained when the pH of the medium is kept in a range of about 5 to about 10, preferably about 6 to about 8.
• the time period for incubation varies depending upon composition, temperature, etc. of the medium but is generally for about 3 to about 10 days. Inoculation of the yeast belonging to the genus Torulopsis producing the esterase at an initial stage of the incubation is effective. Good results may be obtained when inoculation is at the time of initiating the incubation or up to the second day.
• the esterase may be aseptically incorporated prior to the production of the compound (II); alternatively, the culture solution containing the compound (II) may be cyclized to a container retaining the esterase.
• the antibiotic (I) is mainly contained in the culture solution and therefore liquid is removed by centrifugation or filtration and thereafter the active substance is extracted from the filtrate.
• the product can be separated, harvested and purified by means used for the production of ordinary antibiotics utilizing a difference in solubilizing property or solubility in an appropriate solvent, a difference in precipitating property or precipitating rate from a solution, a difference in adsorptive affinity to various adsorbents, a difference in distribution between two liquid phases, etc. These means to be applied singly, in combination of optional orders or repeatedly, if necessary or desired.
• oganomycin E By oxidative deamination-decarbonation of oganomycin E, oganomycin E is converted into 7 ⁇ -(4-carboxybutyrlamido)-3-hydroxymethyl-7 ⁇ -­ methoxy-3-cephem-4-carboxylic acid (hereafter simply referred to as oganomycin GE) which is then purified and recovered.
• oganomycin GE 7 ⁇ -(4-carboxybutyrlamido)-3-hydroxymethyl-7 ⁇ -­ methoxy-3-cephem-4-carboxylic acid
• the aimed compounds such as oganomycin E (or oganomycin GE) are obtained as a free compound as well as a salt such as a usual alkali metal salt (e.g.,Li-, Na-, Ka- salts),an alkali earth metal salt (e.g., Ca-, Mg-, Ba- salts), an organic amine salt (e.g.,triethyl­ amine salt), etc.
• a salt such as a usual alkali metal salt (e.g.,Li-, Na-, Ka- salts),an alkali earth metal salt (e.g., Ca-, Mg-, Ba- salts), an organic amine salt (e.g.,triethyl­ amine salt), etc.
• the effect of the present invention lies in that oganomycin E can be efficiently produced by enzymatically hydrolyzing the 3-side chain ester of cephamycin compound (II).
• cephamycin compound (II) analogous to oganomycin E that is by-produced in the fermentation medium can be immediately converted into oganomycin E in situ so that culture solution containing oganomycin E in a high concentration can be obtained.
• the concentration of oganomycin E produced in the culture solution on the 6th to 7th days during the incubation period may reach 5 times or more that of the conventional process and, the process is more advantageous from an industrial viewpoint.
• Example 1 relates to production of oganomycin E by acting the esterase and cephamycin B and
• Examples 2 and 3 relate to production of oganomycin E by mixing fermentation
• Example 4 relates to utilising a yeast containing esterase.
• Oganomycin GE 7 ⁇ -(4-carboxybutyrlamido)-3-hydroxymethyl-7 ⁇ -­ methoxy-3-cephem-4-carboxylic acid
• Oganomycin GE is a useful compound as an intermediate for producing 7 ⁇ -methoxycephalosporin antibac­ terial compounds.
• cephamycin B was decomposed by 30 to 40% after 7 hours and after 21 hours, by almost 100%; the formation of the corresponding oganomycin E was observed.
• cephamycin B was decomposed by about 70% after 21 hours but the formation of oganomycin E was merely about 13%.
• the culture solution of Torulopsis sp . YE-0807L used herein has a specific ability of decomposing cephamycin B. This decomposition is a reaction for quantitatively accelerating the formation of oganomycin E and this activity is present in the yeast cells but absent in the culture supernatant.
• An Erlenmeymer's flask of 500 ml charged with 50 ml each of a main fermentation medium containing 18% dextrin, 2% glycerine, 3% soybean powders, 2% gluten meal, 0.2% magnesium carbonate and 0.23% sodium hydroxide was prepared. After sterilizing at 120°C for 20 minutes, 2 ml of the above-described seed culture solution was transplanted and, incubation was initiated at 30°C with a rotary shaker at 240 rpm. Separately, a Sakaguchi flask of 500 ml containing 100 ml of GPY medium (1% glucose, 0.25% peptone and 0.25% yeast extract) was sterilized and prepared.
• GPY medium 1% glucose, 0.25% peptone and 0.25% yeast extract
• esterase-producing yeast Torulopsis sp . YE-0807L (FERM BP-1158) was inoculated followed by seed culturing at 30°C for 2 days.
• 1 ml each of the seed culture solution and the esterase-producing yeast was added and the incubation was continued for 7 days.
• 3 flasks were provided for analysis of the concentration of oganomycin E, etc. daily.
• the concentration of the product was determined by HPLC (column: LS224 (made by Toyo Soda Co., Ltd., 4 mm x 500 mm), eluant: 0.02M citric acid (pH 3.2), detection: UV detector 254 nm).
• Example 2 In a fermenter of 30 liters, 20 liters of the fermentation medium of Example 2 were sterilized at 121°C for 30 minutes and 0.8 liters of the seed culture solution of the Y-G19Z strain were transplanted. Fermenta­ tion was initiated at 30°C. On the first day of the fermentation, 100 ml of the seed culture solution of the esterase-producing yeast in GPY medium was transplanted and mixing culture was performed up to the 7th day. Seed culture of both strains was performed in a manner similar to Example 2.
• the desalted matter was adsorbed on 5 liters of Dowex 1 x 2 (Cl ⁇ ) (made by Dow Chemical Co., Ltd.). After washing with water, elution was performed with a 0.2M aqueous salt solution and analysis was performed by HPLC. Fractions of the compound (I) were collected. The eluted fractions were electrically dialyzed for 16 hours using the aforesaid electrodialysis device to effect desalting. After desalting, the system was concentrated under reduced pressure and freeze dried to powder form.
• Dowex 1 x 2 made by Dow Chemical Co., Ltd.
• a fermentation solution was obtained by performing mixing fermentation in a fermenter of 30 liters in a manner similar to Example 2. To the fermentation solution was added 10% of diatomaceous earth (Radiolite #600). After filtering at pH 4, the pH was once changed to 3.0 and immediately thereafter adjusted to 7.0. At this stage, 500 g of wet cells of Trigonopsis variabilis and 50 ml of a 30% hydrogen peroxide were added to the system. The enzyme reaction was performed at 30°C for 1 hour to give 7 ⁇ -(4-­ carboxybutyrylamido)-3-hydroxymethyl-7 ⁇ -methoxy-3-cephem-4-­ carboxylic acid (simply referred to as oganomycin GE).
• oganomycin GE 7 ⁇ -(4-­ carboxybutyrylamido)-3-hydroxymethyl-7 ⁇ -methoxy-3-cephem-4-­ carboxylic acid
• oganomycin GE The formation of oganomycin GE and disappearance of oganomycin E were confirmed by comparing the respective standard specimens under the HPCL conditions described above. After completion of the reaction, the pH was again adjusted to 4 followed by centrifugation and filtration. A clear solution was obtained.
• oganomycin GE solution was passed through a column (2 liters) packed with HP-21 Resin (trademark, made by Mitsubishi Chemical Industry Co., Ltd.) and then passed through a column (8 liters) of SP-207 Resin (trademark, made by Mitsubishi Chemical Industry Co., Ltd.) to adsorb oganomycin GE thereto. After washing with water, fractions containing the product were collected with 25% acetone water. Then, oganomycin GE was adsorbed to Dowex 1 x 2 (2 liters) column (Cl type). After washing with water, 7 liters of the eluate were obtained using a 0.5M triethylamine hydrochloride solution (pH 6.8).
• the oganomycin GE solution was adjusted to pH 6.8 with triethyl amine and then concentrated to dryness. Then, the residue was dissolved in 1.5 liters of methylene chloride to remove insoluble matters and 10 g of activated charcoal powder was added thereto. After stirring for 30 minutes, filtration was carried out. The thus obtained clear solution was concentrated to dryness and further dried in vacuum at room temperature for 48 hours to give 113 g of the powdery product. By comparing with data for the standard specimen in spectrometric analyses by NMR, IR, UV, etc. and HPLC as well as TLC, etc., the thus obtained compound was identified to be oganomycin GE triethyl amine salt. Its purity was about 85%.
• Example 4 shows results obtained by utilizing a microorganism (yeast) carrying the esterase of this invention.
• the culture solution was obtained.
• the cells in the culture solution were subjected to centrifugation, washed with water, subjected to centrifugation again, and collected to obtain the living-cells solution.
• Powders of the living-cells were dispersed in 100% cold-alcohol, and, after 30 minutes, the cells were collected, washed with water to obtain a solution containing un-living cells.
• Each of 5 ml of the living-cells solution or the un-living cells solution was added to a cephamycin B solution (7.3mM, 0.1M phosphoric acid buffer-pH7.2) in a flask, and the mixtures in the flasks were allowed to react by using shaker (30°C, 235 rpm). 0, 4 and 16 hours after the reaction, the reaction solutions were checked by high-­ performance chromatography. The results are shown as compared with control without yeast cells.
• esterase-­ containing materials un-living cells as well as living cells

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• 1986
  • 1986-09-23 ES ES8602118A patent/ES2000401A6/es not_active Expired
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